Transplant rejection occurs when the immune system of the recipient of a transplant, particularly antibodies produced by the recipient, attacks the transplanted organ or tissue. The recipient's immune system recognizes the transplanted organ as foreign tissue and attempts to destroy it. Rejection also occurs when the transplanted organ comprises the donor's lymphocytes or progenitor stem cells, which may generate an immune response to the recipient tissues such as graft vs. host disease. Chronic rejection is a term used to describe all long term loss of function in organ transplants associated with chronic alloreactive immune response. Long term chronic rejection usually leads to a need for a new transplanted organ about a decade after the initial transplant. Human leukocyte antigens (HLA) are one type of molecules within a transplanted organ in which the recipient's immune system attacks that causes a transplant rejection.
An HLA class I molecule consists of a 45-kDa glycoprotein (heavy chain) non-covalently associated with a 12-kDa polypeptide, β2-microglobulin (β2m). Association of β2m with newly synthesized class I heavy chains is required in order for the HLA molecule to transport and present the peptide (Krangel et al., Cell 18: 979, 1979). However, β2m free class I heavy chains were identified on activated T lymphocytes (Schnabl et al., J. Exp. Med. 171:1431, 1990) and other cell surfaces (Bix & Raulet, J. Exp. Med. 176(3) 829-34, 1992). Properly conformed β2m free class I heavy chains were identified on the cells and were believed to have functional importance. β2m can be dissociated from a HLA class I complex on a cell surface by acid treatment (Sugawara et al., J. Immunol. Methods, 100(1-2):83-90, 1987). β2m can also be dissociated from HLA Class I complex coated on microbeads using the similar method of low pH treatment. (Pei et al. Visuals Clinical Histocompatability Workshop 2000, 9-10). Those β2m-free HLA heavy chains are referred to as “denatured antigens.” Antibodies against denatured class I HLA antigens have been detected in human sera, however, they have not been well studied and currently the clinical significance of these antibodies is unclear.
HLA class II molecules are heterodimers formed by noncovalent linkage of two glycosylated polypeptide chains referred to as alpha and beta chains. The α subunit is 33 kDa and the β subunit is 28 kDa, and both chains are transmembrane polypeptides that have the same overall structure. The invariable α chain is encoded by the DRA HLA gene and this chain binds various β chains encoded by the DRB HLA genes. In addition, the DP and Dq HLA gene families each have one gene that encodes an α chain and a β chain. (Reviewed in Choo, Yonsei Med. J. 48: 11-23, 2007).
Cell-based assays are the most widely accepted assay for cross-matching HLA antigens, and these assays are used to determine if a recipient has antibodies in their serum that are cytotoxic to the lymphocytes of a prospective donor. The donor cells for the cross-match assay are collected from either blood or spleen and must be alive, while the recipient serum can be frozen prior to the assay. Exemplary standard procedures for cross-matching include complement-dependent cytotoxicity test (CDC), CDC with antiglobulin augmentation (AHG) and flow Cytometry Cross-match (FC) (Noreen, The American Society for Histocompatibility and Immunogenetics Laboratory Manual, 3rd Ed, I.C.1.1-I.C.1.13).
As the number of identified HLA antigens is continuously increasing, a cell panel must be extremely large to screen for all known HLA antigens in a single assay. In addition, the cell-based assays may be neglecting clinically important antigens as about 50% of transplanted organs will be chronically rejected over a long period of time (known as “long term chronic organ rejection”). Cell-based assays are also limited by the fact that the target cells isolated under the normal condition bear only HLA antigens in their native conformation such as β2m-associated class I antigens (Sugawara et al., J. Immunol. Methods, 100(1-2):83-90, 1987) and heterodimeric class II antigens. Thus, the cell-based assays will not detect antibodies specific for denatured HLA antigens.
The development of solid-phase HLA antibody detection assays, such as bead-based assays, allow for screening of many HLA antigens in one assay. Therefore, many laboratories are using solid-phase HLA antibody detection assays and virtual cross-matching assays in addition to cell-based assays. “Virtual cross-matching” is a procedure that predicts the result of a cell-based cross-match assay. Virtual cross-matching is carried out by comparing the donor antigen profile (donor tissue typing) and the recipient HLA antibody profile as determined by solid-phase assays described herein. For example, some laboratories first screen the recipient sera using a solid-phase assay to determine potential donors matches and subsequently use a cell-based assay for the cross match analysis of the donor cells and recipient sera. With the current screening procedures, sera that is positive for antibodies specific for donor HLA antigens in both the solid-phase assay and the cell-based assay is certain not to be an appropriate match for transplant. However, if the recipient sera is positive for antibodies specific for the donor HLA antibodies in the solid-phase assay but negative in the cell-based assay, there is uncertainty whether this sera is false-positive for HLA-antibodies and whether the tissue can be used for transplant.
One of the advantages of determining the HLA antibody profile using solid-phase assays is that this information can be stored for the transplant recipients. Therefore, biological samples do not need to be acquired at the same time or need to be in the same location for analysis. The HLA antibody profiles will allow for analysis before an organ is shipped for transplant. In addition, solid-phase HLA antigen analysis can screen many more donors and recipient pairs and may screen many more antigens at one time or in fewer assays. In addition, these assays may decrease the incidence in which a positive cross-match organ is shipped for transplant before the determination.
As increasing numbers of sera samples were tested for HLA antibodies, there was a significant increase in the number of samples that were positive using the solid-phase assay but negative using cell-based cross-match assays. In addition, antibodies to denatured class I HLA antigens are known to be present in the human sera and particularly some untransfused males have been shown to have HLA antibodies in their sera.
It is known that acid treated cells, which dissociated βm2 from the surface of HLA class I molecule, can be used to detect antibodies specific for denatured HLA antigens. In addition, panels of microbeads that present denatured class I HLA antigens have been generated and used to detect antibodies specific for these denatured antigens (See Pei et al. Visuals Clinical Histocompatability Workshop 2000, 9-10).
Currently, those of skill in the art do not consider antibodies specific for denatured HLA antigens to induce transplant or transfusion rejections. Generally, the presence of denatured HLA antigens is considered an artifact of sample preparation and detection of antibodies specific for these denatured antigens is not considered a clinically significant step of HLA tissue-typing analysis. One reason for this view is that cell-based assays are the current standard assay for cross-matching, and there are no current assays available for detecting antibodies specific for denatured antigens alone. As virtual cross-matching and solid-phase assays become standard practices in tissue-typing laboratories, there is a need to develop assays that screen for antibodies specific for HLA antigens while determining whether the antibodies are specific for native or denatured antigens. In addition, there is a need to decrease the number of false positive and false negative results of both solid-phase and cell-based tissue typing assays.